Humoral immune responses are critical for protection against pathogens and are a cause of pathogenesis in autoimmune and allergic diseases. Despite their importance, fundamental questions remain regarding how antibody responses are mounted. A major focus of this grant has been to characterize the chemokines and related organizer cues in lymphoid organs that facilitate antibody responses. Key findings pertinent to the current proposal have been: identification of lymph node subcapsular sinus (SCS) macrophages as a site of B cell encounter with opsonized antigen;demonstration that B cells function as antigen transport cells;characterization of CXCR4 and CXCR5 as organizers of the germinal center (GC);visualization of cell migration dynamics in the GC;measurement of B cell -T cell contacts at the follicle-T zone boundary and in the GC leading to evidence that GC B cell selection may occur in part through competition for T cell help. Based on these findings we propose to focus the application on the following three specific aims. One, we seek to further assess how SCS macrophages capture antigen and interact with B cells using fluorescence microscopy, flow cytometry and two-photon imaging approaches. In addition, we will identify chemokine requirements for SCS macrophage positioning using gene expression studies and analysis of gene targeted mice. B cell interaction with follicular dendritic cells will also be visualized. Second we aim to characterize cellular events associated with selection of high affinity B cells in the Germinal Center (GC). We will use adoptive transfer approaches with immunoglobulin `knockin'B cells to study the impact on antibody affinity maturation of chemokine receptor deficiencies that disrupt GC B cell positioning. Third, we will examine the role in the GC response of a further G-protein coupled receptor found to be transcriptionally upregulated in GC B cells. These studies should lead to an improved understanding of how B cells encounter antigen and undergo selection, knowledge that has implications for development of improved vaccines and may suggest novel approaches for reducing unwanted responses to autoantigens or allergens. Public Health Relevance: Humoral immune responses are critical for protection against pathogens and are a cause of pathogenesis in autoimmune and allergic diseases. The proposed studies should lead to an improved understanding of how B cells encounter antigen and undergo selection, knowledge that has implications for development of improved vaccines and may suggest novel approaches for reducing unwanted responses to autoantigens or allergens.